Șef lucr. dr. Văruț Renata Maria [311148]

[anonimizat]. dr. Văruț Ciprian Marius*

Conf. dr. Amzoiu Emilia

Asist. dr. [anonimizat], Department I, Craiova

Corp A’, Str. [anonimizat]. 2, Cod 200349

* [anonimizat]; Tel. [anonimizat]

Abstract

Diabetes is a [anonimizat], immune and endocrine disturbance. [anonimizat] (folium) hydroalcholic extract (20%), comparative with Vaccinum myrtillus (fructus). Diabetes was induced with a single i.p. dose of 180 mg/kg b.w. streptozotocin. [anonimizat], [anonimizat]. [anonimizat]: [anonimizat], IVth consisted  of mice with induced diabetes on therapy with extract of species Vaccinium myrtillus (150 mg/kg b.w), a product recognized for the antioxidant effect. Diabetic mice from IIIrd group was treated with Tragopogon pratensis extract of 150 mg/kg b.w.

[anonimizat]-eosin and analyzed by light microscopy. In the theoretical part of the study we realized with the help of the molecular docking technique the interaction between the polyphenolic compounds and the main factors involved in inflammation and cellular apoptosis in type 1 diabetes mellitus.

[anonimizat]. The theoretical calculus of molecular docking supports the experimental study.

Key words: [anonimizat], [anonimizat]. World Health Organizations and International Diabetes Federations have reported that the number of diabetics in 2000 was 150 million, and by 2025 about 300 million cases are estimated. Of these, 10-12% [anonimizat]. Diabetes induces alterations in activity of enzyme glutathione reductase and glutathione peroxidase. Cell that metabolizes peroxide to water use these enzymes and convert glutathione disulfide back to glutathione []. Alteration of their levels direct the cells to oxidative stress and hence causes injury of cells.

[anonimizat], to peroxide and molecular oxygen. So superoxide is transformed to other compounds which are less toxic [].

Diabetes mellitus determine disturbances in the body lipid profile making the cells more susceptible to lipid peroxidation []. Experimental studies show that free radicals attack polyunsaturated fatty acids in cell membrane due to the presence of multiple bonds [].

The result of lipid peroxidation is malondialdehyde (MDA) that can be used to measure lipid peroxides in reaction with thiobarbituric acid [].

In many studies, oxidative stress has been shown to contribute to the progression of diabetes, so it plays an important role in diabetes, causing damage to insulin action and increased incidence of complications.

The existance of high levels of polyphenols in plant products is a prerequisite for favorable therapeutic effects in dysfunctions present in diabetes. Long term consumption of diets rich in plant polyphenols have positive antioxidant effects on the human body, reducing de development risk of cancers, cardiovascular diseases, diabetes, osteoporosis and neurodegenerative diseases. The most important compounds are classified in phenolic acids, flavonoids, stilbenes and lignans [].

The study of the effects of natural extracts on cellular and molecular mechanisms involved in diabetes is a priority area of current research. Computational methods are used extensively in recent years to study the formation of intermolecular complexes between the ligand and the biological receptor. Such a method, referred to as "Molecular docking", enables prediction of favorable orientations of a molecule (ligand) with a second molecule (receptor) by linking them so as to form a stable complex [, ]. In other words, the method can be used to predict the binding affinity between the two molecules []. A low negative energy indicates the stability of the system []. This technique is also used in the present study to test polyphenolic compounds (chlorogenic acid, rutoside and apigenol) as potential antioxidants involved in inflammatory processes and cellular apoptosis in type 1 diabetes mellitus (TNF-alpha, IL-6, COX- 2).

Tragopogon pratensis L. subsp pratensis – goat beard, Asteraceae is a biannual plant, found in Europe and North America, growing in the plain and on the roadside. It is commonly found in England, the southeastern part of Scotland and central Ireland. The chemical composition of the plant has been elucidated by spectral and chemical techniques and contains nine triterpenic saponosides, called tragoponoids A-I, found throughout the plant structure, along with five other triterpenic glycosides [].

The fresh roots of the plant were traditionally used for the preparation of dietary salads, recommended for obese or diabetic persons. In Armenia, kids made gum from the juice of the plant, which was collected and dried in glass pots [, ].

Tragopogon pratensis is used to treat liver and gall bladder disease. It has been observed that the extract possesses detoxifying properties and stimulates appetite and digestion. Due to the high inulin content it can be consumed by diabetics. The root is astringent, diuretic, depurative, expectorant and stomachic. Recent studies have shown that the methanolic extract exhibits antiproliferative and tumor growth inhibiting properties [].

The aim of the paper is to test the content of polyphenolcarboxylic acids and flavonoids for Tragopogon pratensis extract, antioxidant capacity, regenerative pancreatic and hepatic effects, taking reference to the effects of cranberry fruits. It also aims to correlate the experimental result with the theoretical docking analysis. "Molecular docking" technique is an effective tool for selecting potential therapeutic agents.

Material and method

Preparation of tinctures by the simple percolating method (F.R. X)

Plant material. Fresh herba of T. pratensis were collected in the city of Craiova, Romania and V. myrtillus was bought as fresh fruits from a supermarket. Both plant samples were air dried in the shade, at ambient temperature. Herbarium voucher samples are deposited in the Laboratory of Pharmacognosy, Faculty of Pharmacy, University of Medicine and Pharmacy of Craiova, Romania. Preparing the sample Vegetable product was used as tinctures, manufactured by simple leaching [], in a ratio of vegetable / solvent (ethanol 70c ) of 1:5 (F.R. X). For each gram of plant product, 0.5 ml of dilute alcohol was used for wetting, mixed and left at room temperature for three hours in tightly closed containers. The vegetable products were passed through a 1st sieve and introduced into the percolator by light pressing; the solvent was gradually added until it started to flow through the lower tap left open beforehand and above the mixture there was still a layer of liquid [, ].

The valve was closed and left to stand for 24 hours, then start the actual percolation. The percolation speed must be adjusted so that in 24 hours 1.5 g of the extraction solution is obtained per gram of plant product. Throughout the extraction, for two weeks, the plant products were coated with the solvent. Percolation was discontinued when 500 ml of each tincture was obtained. After standing for seven days at 5-10° C, tinctures were filtered and divided into a brown glass container with volume of 100 ml, maintained tightly closed, protected from light, at room temperature [].

Determination of antioxidant capacity of tinctures

Determination of the total polyphenol content

Substances and reagents are Sigma-Aldrich products (Germany): gallic acid (GAE), Folin-Ciocalteu reagent, sodium bicarbonate, aluminum chloride, potassium acetate, quercetol.

A calibration curve of gallic acid in the range of 0 to 1500 mg / l was constructed as follows: 150 mg of gallic acid were dissolved in a 100 ml volumetric flask, initially adding 10-20 ml of 98 % ethanol until complete dissolution of the substance and completed to the mark with distilled water. From the obtained solution were added 0, 1, 2, 3, 5, 10 and 20 ml in 100 ml volumetric flasks and made up to the mark with distilled water. From both the samples and the calibration solutions, 200 μl were added, over which 2.5 ml of Folin-Ciocalteu reagent diluted in 1:10 with distilled water was added. Samples were left for four minutes at room temperature, then 2 ml of 75 g / l sodium bicarbonate solution were added. The mixture thus obtained was left in the dark for two hours. Absorbance was measured using an Able-Jasco V530 UV / VIZ spectrophotometer at the wavelength of 765 nm. The obtained results were expressed in g equivalents of gallic / liter of tincture [g GAE / l]. All measurements were made in triplicate, calculating the mean ± standard deviation.

Determination of the total flavonoid

The flavonoid content was determined colorimetrically by the aluminum chloride method. Thus, 0.5 ml of each tincture was used with 0.1 ml of 10% aluminum chloride, 0.1 ml of 1M potassium acetate and 2.8 ml of distilled water. The mixture was left for a few minutes at room temperature and then spectrophotometer at 415 nm. Was built a calibration curve having standard quercetol (QE), with concentrations ranging from 0-200 mg / l.

Determination of total carotenoid, chlorophyll a and chlorophyll b

Diluted tinctures ranging from 1-4 mg / ml using extraction solvent (ethanol 70c) were analyzed on a Jasco-Able 530 UV / VIZ spectrophotometer at three different wavelengths (470, 653 and 666 nm), determining absorption (Abs). The concentrations of carotenoids, chlorophyll (Ca) and chlorophyll b (Cb) were determined [] based on the formulations proposed by Lichtenthaler and Wellburn (1983), as follows:

▪ Carotenoid total [mg / l] = 1000Abs470 – 2.860Ca – 129.2Cb / 245;

▪ Chlorophylla a [mg / l] = 15.65Abs666 – 7.340Abs653;

▪ Chlorophyll b [mg / l] = 27.05Abs653 – 11.21Abs666.

Testing in vivo antioxidant potential of plant extracts, determining the level of activity of superoxide dismutase, glutathione peroxidase, glutathione reductase, and levels of reactive substances with thiobarbituric acid

The protocol for inducing diabetes mellitus

The animals were kept fasting with free access to water for 12 hours before and three hours after the injection of streptozotocin injection. For the induction of experimental diabetes, streptozotocin was injected at a single dose of 180 mg / kg, intraperitoneally, the injected amounts being determined according to the body weight of the animal;

Animals with blood glucose values above 300 mg / dl, confirmed by two determinations (three days and one week after streptozotocin injection), were considered diabetic and were introduced into the research groups.

The experiment was performed on four groups of five mice, with the animals receiving the prescribed medication daily at the same time for five weeks. Three control groups were used in the experiment: group I, group II and group IV.

Group I: The healthy, control mice, consisting of mice with normal pancreatic and hepatic function, did not receive medication.

Group II: The diabetic control group, consisting of diabetic mice, did not receive medication.

Group III: consisting of diabetic mice, received by gavage Tragopogon pratensis tincture 20% of folium 150 mg / kg-body every day, once a day, for five weeks.

Group IV: Diabetics consisting of mice, received by gavage tincture 20% of Myrtilli fructus, 150 mg / kg-body every day, once a day, for five weeks.

For quantitative in vitro quantification of antioxidant activity, kits produced by Randox Laboratories were used. The measurement method is colorimetric, using a Beckman UV-VIS spectrophotometer, DU-65 model.

Hemolysis of red blood cells: 0.5 ml of whole blood was centrifuged for 10 min. at 3000 rpm, the plasma was aspirated, and the cells washed four times with physiological solution and centrifuged. The hemolysis was produced with 2 ml of cold bidystiled water and left at rest for 15 minutes at 40° C. Dilution was performed with 0.01 M phosphate buffer pH 7 and used to evaluate the activities of antioxidant enzymes.

Evaluation of superoxide dismutase activity (SOD). The erythrocytic SOD activity was measured by the inhibition rate of 2- (4-iodophenyl) -3 (4-nitrophenol) -5-phenyltetrazolium (INT) oxidation by xanthine oxidase reaction superoxide (RanSOD kit).

Evaluation of glutathione peroxidase activity (GPx). GPX erythrocyte activity was measured from the absorbance variation at 340 nm after oxidation of GSH by cumene hydroperoxide and reduction of GSSG by GR and NADPH, H + (Ransel kit).

Evaluation of glutathione reductase activity (GR). GR activity was measured from the decrease in absorbance at 340 nm after oxidation of NADPH, H + with the reduction of GSSG to GSH (Randox GR kit).

Evaluation of lipid peroxides (POL). Malondialdehyde, the major lipid peroxidation product, reacts with 20% thiobarbituric acid to form a compound that absorbs at 532 nm.

Histological analysis

Histological tissue analysis was performed to observe the correlation between the antioxidant effects of plant extracts and the reparative tissue potential on the organs.

After five weeks of plant extracts testing, the animals were sacrificed, tissues of interest were removed and processed according to the protocol, achieved smears stained with hematoxylin-eosin and analyzed in the light microscope. From each batch we collected samples from the pancreas and liver.

Starting from the tissues harvested from the test animals, several steps were taken to obtain the smear to be examined, namely the paraffin inclusion technique and the histological staining technique with hematoxylin eosin.

Coloring with hematoxylin-eosin mixture gives the possibility of recognizing tissues due to the different coloring of their components: the nucleus of the cells appears intensely colored in blue-violet, the cytoplasm appears colored with light violet, the collagen fibers are pale pink, the elastic fibers and the reticulin is highlighted [].

Docking molecular

For the modeling and optimization of ligands, the Avogadro program was used (PM3 semi-empirical modeling) []. The target proteins were downloaded from the Protein Data Bank database [] and optimized with the Modrefiner software []. The molecular docking analysis was performed using the Autodock 4.2 software, the molecules being visualized using Pymol [].

Results and discussions

The -OH group in the polyphenols constituent is responsible for the antioxidant character, canceling the effect of free radicals such as peroxo, oxo, hydroxo.

Table 1 – Results of determination of antioxidant capacity of tinctures

The determinations showed that both tinctures contain appreciable amounts of polyphenols (Table 1), the highest content being the tincture of cranberry fruits (M-fr, 0.771 ± 0.087 mg GAE / l). For both tinctures, the amount exceeds the total polyphenol content, a possible explanation being that tinctures have a lower concentration of polyphenolic acids compared to flavonoids. The highest amount of carotenoids and chlorophylls is contained in M-fr tincture.

The plasma level of glutathione reductase is low in the presence of diabetes mellitus and oxidative stress (Table 2) [].

Table 2 – Values of the level of antioxidant enzymes and lipid peroxides in the studied groups

M = mean; SD = standard deviation

At the end of the experiment, untreated diabetic group II had the lowest mean glutathione reductase 46.3 ± 2.12 U / ml. Myrtilli fructus (M-fr) tincture has antioxidant properties noted by the mean glutathione reductase enzyme of 52.5 ± 0.70 U / ml, higher than that of batch II, 46.3 ± 2.12 U / ml. The tincture of Tragoponis pratensis folium (TPF) has significant antioxidant efficacy and its mean values are 60.3 ± 8.63 U / ml.

At the end of the experiment, the untreated diabetic group II had the lowest mean glutathione peroxidase 3415.5 ± 98.29 U / l. The Myrtilli fructus tincture has antioxidant properties, resulting from the comparison of the mean value of the glutathione peroxidase group IV treated with it 4 301 ± 11.31 U / l, with the average values of the batch II (3415.5 ± 98.29 U / l) and batch I (4251.5 ± 28.99 U / l). Tragoponis pratensis folium tincture has superior anti-oxidant therapeutic potency M-fr, as measured by mean plasma glutathione peroxidase levels of 5652 ± 1370.37 U / l.

Supplements containing antioxidants, including SOD-mimetics, reduce ROS concentration, and increase the level of antioxidant enzymes, being effective in preventing diabetes [].

The highest superoxide dismutase level was obtained in lot III, treated with 20% vegetable extract, 150 mg / kg body weight, from Tragopogon pratensis, having values higher than group I, healthy (203.8 ± 1.84) , lot II (191.7 ± 10.75 U / ml), lot IV (194.15 ± 3.18 U / ml).

Lipid peroxidation produces highly reactive aldehydes: MDA, acrolein, 4-hydroxynonenal (HNE), 4-oxononenal (UN), and isolevuglandin (IsoLGs). MDA is considered a primary biomarker of free radical damage and the presence of oxidative stress. In diabetes, significantly higher levels of thiobarbituric acid (TBARS) reactive substances were observed in red blood cells as well as serum and decreased antioxidant enzyme activity. Recently, a clinical study by Bandeira and colleagues (2012) notes a correlation between lipid peroxidation, hyperglycemia, HbA1c level and oxidative stress in diabetes []. The highest value of lipid peroxides was observed in lots II, III and IV having considerably lower values.

In the literature, histological analysis of the pancreas of laboratory animals (mice, rats) with streptozotocin diabetes reveals a reduction in the number of Langerhans islets, degeneration, altered form and aggregation of beta-pancreatic cells, hydropathic degeneration, and necrosis [].

In our study, compared with group I, the pancreas of the animals in the second group exhibited important structural changes, represented by the necrotic-haemorrhagic lesions (Figure 1).

Lots III and IV, consisting of diabetic mice, each treated with a plant extract, exhibited variable changes in the pancreatic structure. Through histological analysis, were observed from architectural preservation to hyalinization of acins, dystrophic lesions, necrotic haemorrhagic lesions.

Histological analysis allowed structural hepatic changes to be observed in the different test batches (Figure 2). In group II, structural alterations of parenchyma and stroma were observed.

Lots III and IV show variable hepatic structural changes ranging from granular dystrophic changes to granulo-vacuolar dystrophy to cytohepatonecrosis.

Molecular docking

Figure 3 shows optimized structures using the Avogadro program for the three polyphenolic compounds.

Figure 3 – Optimized structures using the Avogadro program for chlorogenic acid, rutoside and apigenol (from left to right).

For a better "docking", the program uses LGA, which is a combination of the genetic and local search algorithms, allowing for the acceleration of search for the most favorable docking orientations in the receptor molecule with the translation and rotation of the ligand to obtain optimal guidelines.

Table 3 shows the values of binding energies (affinities) between ligands and the biological receptor.

Table 3 – Values of binding energies in ligand-receptor biological ligands

From the data presented in Table 3, it can be concluded that each of the three compounds that enter the plant extract of Tragopogon pratensis contributes to the antioxidant capacity manifested. Therefore, the theoretical data obtained with the "Molecular Docking" technique is in agreement with the experimental ones.

Conclusions

Diabetic chemical agents (aloxane, streptozotocin) destroy beta-pancreatic cells by various mechanisms and induce DZ type 1 with the synthesis of islet anticylic autoantibodies.

Both plant extracts tested containes antioxidant therapeutic efficacy due to the polyphenolic compounds, limiting the destructive effects of streptozotocin.

Following the study, we can see that the high glucose level, sustained for a long time, induces oxidative stress, which is reflected by the increased concentration of malondialdehyde and low antioxidant enzymes. Tragopogon pratensis demonstrates a strong antioxidant effect, the third group treated with it, showing the highest values of enzymatic activity for glutathione reductase, glutathione peroxidase and superoxide dismutase.

In our study, compared with group I, the pancreas of the animals in the second group exhibited important structural changes, represented by the necrotic-haemorrhagic lesions. Lots III and IV, consisting of diabetic mice, each treated with a plant extract, exhibited variable changes in the pancreatic structure by histological analysis, observing the architecture retained in acin hyalinization, dystrophic lesions, necrotic haemorrhagic lesions.

Microscopic observations have highlighted structural liver abnormalities in diabetic mice as compared to normal mice. In group II, structural alterations of parenchyma and stroma were observed. Lots III and IV show structural changes in varying proportions ranging from granular dystrophic changes to granulo-vacuolate dystrophy to cytohepatonecrosis.

The molecular docking explains the efficacy of plant extracts through the polyphenol-cytokine / enzyme binding energy values.The theoretical calculus of molecular docking supports the experimental study.

"Molecular docking" technique is an effective tool for selecting potential therapeutic agents.

References